Apparatus and method for a nanocalorimeter for detecting chemical reactions

ABSTRACT

A nanocalorimeter array for detecting chemical reactions includes at least one thermal isolation region residing on a substrate. Each thermal isolation region includes at least one thermal equilibration region, within which resides a thermal measurement device connected to detection electronics.

CROSS REFERENCE TO RELATED APPLICATIONS

The following copending application, U.S. application Ser. No.10/115,336, filed Mar. 29, 2002, titled “Apparatus and Method for UsingElectrostatic Force to Cause Fluid Movement”, is assigned to the sameassignee of the present application. The entire disclosure of thiscopending application is totally incorporated herein by reference in itsentirety.

INCORPORATION BY REFERENCE

The following U.S. patents are fully incorporated herein by reference:U.S. Pat. No. 5,967,659 (“Ultrasensitive Differential Microcalorimeterwith User-selected Gain Setting” to Plotnikov et al.); U.S. Pat. No.6,079,873 (“Micron-scale Differential Scanning Calorimeter on a Chip” toCavicchi et al.); U.S. Pat. No. 6,096,559 “Micromechanical CalorimetricSensor” to Thundat et al.); and U.S. Pat. No. 6,193,413 (“System andMethod for an Improved Calorimeter for Determining ThermodynamicProperties of Chemical and Biological Reactions” to Lieberman).

BACKGROUND OF THE INVENTION

This invention relates generally to an apparatus and method for animproved nanocalorimeter, and more specifically, to a system and methodfor an improved nanocalorimeter for measuring the heat released orabsorbed during chemical reactions.

Calorimetry is used to measure enthalpic changes, including enthaplicchanges arising from reactions, phase changes, changes in molecularconformation, temperature variations, and other variations of interestthat may occur for a particular specimen. By measuring enthalpic changesover a series of conditions, other thermodynamic variables may bededuced. For example, measurements of enthalpy as a function oftemperature reveal the heat capacity of a specimen, and titrations ofreacting components can be used to deduce the binding constant andeffective stoichiometry for a reaction. Calorimetry measurements areuseful in a broad variety of applications, including, for example,pharmaceuticals (drug discovery, decomposition reactions,crystallization measurements), biology (cell metabolism, druginteractions, fermentation, photosynthesis), catalysts (biological,organic, or inorganic), electrochemical reactions (such as in batteriesor fuel cells), and polymer synthesis and characterization, to name afew. In general, calorimetry measurements can be useful in the discoveryand development of new chemicals and materials of many types, as well asin the monitoring of chemical processes. Standard calorimeters requirerelatively large samples (typically about 0.5 ml to 10 liters) andusually measure one sample at a time. As such, these systems cannot beused to measure very small samples, as might be desired for precious orhighly reactive materials. Furthermore, standard calorimeters cannot beused effectively to monitor a large number of reactions of small samplesize in parallel, as is required in order to perform studies usingcombinatorial chemistry techniques.

In recent years, researchers and companies have turned to combinatorialmethods and techniques for discovering and developing new compounds,materials, and chemistries. For example, pharmaceutical researchers haveturned to combinatorial libraries as sources of new lead compounds fordrug discovery. As another example, Symyx Technologies® is applyingcombinatorial techniques to materials discovery in the life sciences,chemical, and electronics industries. Consequently, there is a need fortools that can measure reactions and interactions of large numbers ofsmall samples in parallel, consistent with the needs of combinatorialdiscovery techniques. Preferably, users desire that these tools enableinexpensive measurements and minimize contamination andcross-contamination problems.

In some cases, the sample to be studied is precious, and it might not beacceptable to use the relatively large amount of material required by astandard microcalorimeter to perform only one measurement. For example,one may desire to study a natural extract or synthesized compound forbiological interactions, but in some cases the available amount ofmaterial at concentrations large enough for calorimetry might be no morethan a few milliliters. Performing a measurement in standardmicrocalorimeters, such as those sold, for example, by MicroCal® Inc.(model VP-ITC) or Calorimetry Sciences Corporation® (model CSC4500),requires about 1 ml of sample, which means that one would possibly beFaced with using a majority or all of the precious material for one or asmall series of measurements. Tools that enable calorimetricmeasurements with much smaller sample sizes would be helpful inovercoming this limitation.

One of the most popular uses of combinatorial techniques to date hasbeen in pharmaceutical research. Pharmaceutical researchers have turnedto combinatorial libraries as sources of new lead compounds for drugdiscovery. A combinatorial library is a collection of chemical compoundswhich have been generated, by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” asreagents. For example, a combinatorial polypeptide library is formed bycombining a set of amino acids in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptidecompound). Millions of chemical compounds can theoretically besynthesized through such combinatorial mixing of chemical buildingblocks.

Once a library has been constructed, it must be screened to identifycompounds, which possess some kind of biological or pharmacologicalactivity. For example, screening can be done with a specific biologicalcompound, often referred to as a target, that participates in a knownbiological pathway or is involved in some regulation function. Thelibrary compounds that are found to react with the targets arecandidates for affecting the biological activity of the target, andhence a candidate for a therapeutic agent.

Through the years, the pharmaceutical industry has increasingly reliedon high throughput screening (HTS) of libraries of chemical compounds tofind drug candidates. HTS describes a method where many discretecompounds are tested in parallel so that large numbers of test compoundsare screened for biological activity simultaneously or nearlysimultaneously. Currently, the most widely established techniquesutilize 96-well microtitre plates. In this format, 96 independent testsare performed simultaneously on a single 8 cm×12 cm plastic plate thatcontains 96 reaction wells. These wells typically require assay volumesthat range from 50 to 500 μl. In addition to the plates, manyinstruments, materials, pipettors, robotics, plate washers and platereaders are commercially available to fit the 96-well format to a widerange of homogeneous and heterogeneous assays. To achieve fastertesting, the industry is evolving to plates that contain 384 and 1536wells.

A variety of measurement approaches has been used to screencombinatorial libraries for lead compounds, one of which is theinhibitor assay. In the inhibitor assay, a marker ligand, often thenatural ligand in a biological pathway, is identified that will bindwell with the target protein molecule. The assay requires the chemicalattachment of a fluorescent molecule to this marker ligand such that thefluorescent molecule does not affect the manner in which the markerligand reacts with the target protein. To operate an inhibitor assay,the target protein is exposed to the test ligands in microtitre wells.After a time necessary for reaction of the test ligand to the targetprotein, the marker ligand is applied. After a time for reaction withthe marker ligand, the wells are rinsed such that non-reacted markerligand is washed away. In wells where the target protein and the testligand have reacted, the test ligand blocks the active site of thetarget protein so the marker ligand cannot react and is washed away,while in cells where the target protein and test ligand have notreacted, the marker ligand reacts with the target protein and is notwashed away. By investigating the wells for the presence of fluorescenceafter the washing, reactions of test ligands and target proteins can bedetermined as having occurred in wells where no fluorescence isobservable.

However, the inhibitor assay requires time and expense to develop theassay. The principal components that need development are discovering amarker ligand and attaching a fluorophore to the marker in a manner thatdoes not affect its reaction with the target protein. Attaching thefluorescent marker can often take 3 months of development or more andcost $250 k or more once the marker ligand is identified. An assaymethod that avoids such assay development, such as measuring the heat ofthe reaction with calorimetry, would eliminate this cost and lime delayin the discovery process.

Calorimetry measurements are commonly utilized in biophysical andbiochemical studies to determine energy changes as indications ofbiochemical reactions in a media. Prior techniques for measurementsinclude using electrodes, thermopiles, optical techniques, andmicrocalorimeters for measurements within a sampled media. There is agreat interest in developing ultra-miniature microcalorimeter devicesthat require very small volumes of sampled media for accurate detectionand measuring of biochemical reactions on, or in close proximity to, themicrocalorimeter and which can be applied in a manner to quickly measurelarge numbers of reactions such that it can be as efficient as assayssuch as inhibitor assays which can be used in HTS to screen perhaps100,000 test ligands a day.

The following disclosures may be relevant and/or helpful in providing anunderstanding of some aspect of the present invention:

In Plotnikov et al., U.S. Pat. No. 5,967,659 (“UltrasensitiveDifferential Microcalorimeter with User-selected Gain Setting”), adifferential calorimeter is disclosed that includes sample and referencecells, a thermal shield surrounding the cells, heating devices thermallycoupled to the thermal shield and the cells, a temperature monitoringsystem, and a control system. The temperature monitoring system monitorsthe temperature of the shield, cell temperatures, and temperaturedifferentials between the cells and the shield. The control systemgenerates output signals for control of the heating devices, with a gainsetting and scan rate selected by means of a user interface. Outputcontrol signals are functions of input temperature signals and theuser-selected gain setting, as well as functions of input temperaturesignals and the user-selected scan rate using a mapping function storedin memory.

In Cavicchi et al., U.S. Pat. No. 6,079,873 (“Micron-scale DifferentialScanning Calorimeter on a Chip”), a differential scanningmicrocalorimeter produced on a silicon chip enables microscopic scanningcalorimetry measurements of small samples and thin films. The chip,fabricated using standard CMOS processes, includes a reference zone anda sample zone. The reference and sample zones may be at opposite ends ofa suspended platform or may reside on separate platforms. Each zone isheated with an integrated polysilicon heater. A thermopile consisting ofa succession of thermocouple junctions generates a voltage representingthe temperature difference between the reference and sample zones.

In Thundat et al., U.S. Pat. No. 6,096,559 (“MicromechanicalCalorimetric Sensor”), a calorimeter sensor apparatus utilizesmicrocantilevered spring elements for detecting thermal changes within asample containing biomolecules which undergo chemical and biochemicalreactions. The spring element includes a bimaterial layer of chemicalson a coated region on at least one surface of the microcantilever. Thechemicals generate a differential thermal stress across the surface uponreaction of the chemicals with an analyte or biomolecules within thesample due to the heat of chemical reactions in the sample placed on thecoated region. The thermal stress across the spring element surfacecreates mechanical bending of the microcantilever. The spring elementhas a low thermal mass to allow detection and measuring of heattransfers associated with chemical and biochemical reactions within asample place on or near the coated region. Deflections of the cantileverare detected by a variety of detection techniques.

In Lieberman, U.S. Pat. No. 6,193,413 (“System and Method for anImproved Calorimeter for Determining Thermodynamic Properties ofChemical and Biological Reactions”) a microcalorimeter includes a thinamorphous membrane anchored to a frame within an environmental chamber.Thermometers and heaters are placed on one side of a thermal conductionlayer mounted on the central portion of the membrane. Samples are placedon two such membranes; each sample is heated and its individual heatcapacity determined. The samples are then mixed by sandwiching the twomicrocalorimeters together to cause a binding reaction to occur. Theamount of heat liberated during the reaction is measured to determinethe enthalpy of binding.

SUMMARY OF THE INVENTION

Briefly stated, and in accordance with one aspect of the presentinvention, there is disclosed a nanocalorimeter array for detectingchemical reactions. The nanocalorimeter array includes at least onethermal isolation region residing on a substrate. Each thermal isolationregion includes at least one thermal equilibration region, within whichresides a thermal measurement device connected to detection electronics.

In accordance with another aspect of the present invention, there isdisclosed a method for detecting chemical reactions at lowconcentrations through the use of a nanocalorimeter. Drops ofpotentially reactive chemical solutions are deposited within a thermalequilibration region of the nanocalorimeter and the drops are thenmerged. Thermal change occurring within the merged drops is measured toidentify chemical solutions which cause thermal change.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of the instant invention will beapparent and easily understood from a further reading of thespecification, claims and by reference to the accompanying drawings inwhich:

FIG. 1 is a block diagram depicting components of a nanocalorimeter inaccordance with one embodiment of the present invention;

FIG. 2 is a block diagram depicting components of a nanocalorimeter inaccordance with another embodiment of the present invention;

FIG. 3 is a block diagram depicting components of a nanocalorimeter inaccordance with yet another embodiment of the present invention;

FIG. 4 is a block diagram depicting components of a nanocalorimeter inaccordance with yet another embodiment of the present invention;

FIG. 5 is a diagram of the arrangement of the measurement thermometersand reference thermometers according to the present invention;

FIG. 6 is an illustration of one method for merging of deposited dropsaccording to the present invention;

FIG. 7 is a schematic of the electronic measuring system in accordancewith the system and method of the present invention;

FIG. 8 is a block diagram depicting an array of components of ananocalorimeter in accordance with another embodiment of the presentinvention;

FIG. 9 is a cross-sectional diagram illustrating the operatingenvironment of the nanocalorimeter in accordance with an embodiment ofthe present invention;

FIG. 10 is a cross-sectional diagram illustrating one embodiment of theprocess flow of the nanocalorimeter in accordance with the presentinvention; and

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “ligand” refers to an agent that binds a targetmolecule. In the case in which the target molecule is a target protein,the agent may bind the target protein when the target protein is in itsnative conformation, or when it is partially or totally unfolded ordenatured. According to the present invention, a ligand is not limitedto an agent that binds a recognized functional region of the targetprotein e.g. the active site of an enzyme, the antigen-combining site ofan antibody, the hormone-binding site of a receptor, a cofactor-bindingsite, and the like. In practicing the present invention, a ligand canalso be an agent that binds any surface or internal sequences orconformational domains of the target protein. Therefore, the ligands ofthe present invention encompass agents that in and of themselves mayhave no apparent biological function, beyond their ability to bind tothe target protein in the manner described above.

As used herein, the term “test ligand” refers to an agent, comprising acompound, molecule or complex, which is being tested for its ability tobind to a target molecule. Test ligands can be virtually any agent,including without limitation metals, peptides, proteins, lipids,polysaccharides, nucleic acids, small organic molecules, andcombinations thereof. Complex mixtures of substances such as naturalproduct extracts, which may include more than one test ligand, can alsobe tested, and the component that binds the target molecule can bepurified from the mixture in a subsequent step.

As used herein, the term “target protein” refers to a peptide, proteinor protein complex for which identification of a ligand or bindingpartner is desired. Target proteins include without limitation peptidesor proteins known or believed to be involved in the etiology of a givendisease, condition or pathophysiological state, or in the regulation ofphysiological function. Target proteins may be derived from any livingorganism, such as a vertebrate, particularly a mammal and even moreparticularly a human. For use in the present invention, it is notnecessary that the protein's biochemical function be specificallyidentified. Target proteins include without limitation receptors,enzymes, oncogene products, tumor suppressor gene products, vitalproteins, and transcription factors, either in purified form or as partof a complex mixture of proteins and other compounds. Furthermore,target proteins may comprise wild type proteins, or, alternatively,mutant or variant proteins, including those with altered stability,activity, or other variant properties, or hybrid proteins to whichforeign amino acid sequences, e.g. sequences that facilitatepurification, have been added.

As used herein, “test combination” refers to the combination of a testligand and a target protein. “Control combination” refers to the targetprotein in the absence of a test ligand.

As used herein, “screening” refers to the testing of a multiplicity ofmolecules or compounds for their ability to bind to a target molecule.

As used herein, the term “lead molecule” refers to a molecule orcompound, from a combinatorial library, which displays relatively highaffinity for a target molecule. High affinity is detected by thisinvention through the heat released in a chemical reaction. The terms“lead compound” and “lead molecule” are synonymous.

As used herein, the term “target molecule” encompasses peptides,proteins, nucleic-acids, and other receptors. The term encompasses bothenzymes and proteins which are not enzymes. The term encompassesmonomeric and multimeric proteins. Multimeric proteins may be homomericor heteromeric. The term encompasses nucleic acids comprising at leasttwo nucleotides, such as oligonucleotides. Nucleic acids can besingle-stranded, double-stranded, or triple-stranded. The termencompasses a nucleic acid which is a synthetic oligonucleotide, aportion of a recombinant DNA molecule, or a portion of chromosomal DNA.The term target molecule also encompasses portions of peptides,secondary, tertiary, or quaternary structure through folding, withsubstituents including, but not limited to, cofactors, coenzymes,prosthetic groups, lipids, oligosaccharides, or phosphate groups.

As used herein, the term “molecule” refers to the compound, which istested for binding affinity for the target molecule. This termencompasses chemical compounds of any structure, including, but notlimited to nucleic acids and peptides. More specifically, the term“molecule” encompasses compounds in a compound or a combinatoriallibrary. The terms “molecule” and “ligand” are synonymous.

As used herein, the term “thermal change” encompasses the release ofenergy in the form of heat or the absorption of energy in the form ofheat.

As used herein, the term “contacting a target molecule” refers broadlyto placing the target molecule in solution with the molecule to bescreened for binding. Less broadly, contacting refers to the turning,swirling, shaking or vibrating of a solution of the target molecule andthe molecule to be screened for binding. More specifically, contactingrefers to the mixing of the target molecule with the molecule to betested for binding. Mixing can be accomplished, for example, by repeateduptake and discharge through a pipette tip or by deposition by roboticmeans. Preferably, contacting refers to the equilibration of bindingbetween the target molecule and the molecule to be tested for binding.

As used herein, the term “biochemical conditions” encompasses anycomponent, thermodynamic property, or kinetic property of a physical,chemical, or biochemical reaction. Specifically, the term refers toconditions of temperature, pressure, protein concentration, pH, ionicstrength, salt concentration, time, electric current, potentialdifference, and concentrations of cofactor, coenzyme, oxidizing agents,reducing agents, detergents, metal ion, ligands, buffer components,co-solvents including DMSO (dimethyl sulfoxide), glycerol, and relatedcompounds, enhancers, and inhibitors.

The present invention encompasses nanocalorimeters and nanocalorimeterarrays that enable measurement of enthalpic changes, such as enthalpicchanges arising from reactions, phase changes, changes in molecularconformation, and the like. Furthermore, the present inventionencompasses combinatorial methods and high-throughput screening methodsthat use nanocalorimeters in the study, discovery, and development ofnew compounds, materials, chemistries, and chemical processes, as wellas high-throughput monitoring of compounds or materials, orhigh-throughput monitoring of the processes used to synthesize or modifycompounds or materials. The present invention also relates to compoundsor materials identified by the above methods and their therapeutic uses(for diagnostic, preventive or treatment purposes), uses in purificationand separation methods, and uses related to their novel physical orchemical properties. For the purposes herein, a nanocalorimeter refersto a device capable of measuring heats of reaction in the range ofnanocalories.

As an example, the present invention encompasses high-throughputscreening methods for identifying a ligand that binds a target protein.If the target protein to which the test ligand binds is associated withor causative of a disease or condition, the ligand may be useful fordiagnosing, preventing or treating the disease or condition. A ligandidentified by the present method can also be one that is used in apurification or separation method, such as a method that results inpurification or separation of the target protein from a mixture. Thepresent invention also relates to ligands identified by the presentmethod and their therapeutic uses (for diagnostic, preventive ortreatment purposes) and uses in purification and separation methods.

An important feature of the present invention is that it will detect anycompound that binds to any sequence or domain of the target protein, notonly to sequences or domains that are intimately involved in abiological activity or function. The binding sequence, region, or domainmay be present on the surface of the target protein when it is in itsfolded state, or may be buried in the interior of the protein. Somebinding sites may only become accessible to ligand binding when theprotein is partially or totally unfolded.

In practicing the present invention, the test ligand is combined with atarget protein, and the mixture is maintained under appropriateconditions and for a sufficient time to allow binding of the test ligandto the target protein. Experimental conditions are determinedempirically for each target protein. When testing multiple test ligands,incubation conditions are usually chosen so that most ligand:targetprotein interactions would be expected to proceed to completion. Inhigh-throughput screening applications, the test ligand is usuallypresent in molar excess relative to the target protein. The targetprotein can be in a soluble form, or, alternatively, can be bound to asolid phase matrix. The matrix may comprise without limitation beads,membrane filters, plastic surfaces, or other suitable solid supports.

Binding to a given protein is a prerequisite for pharmaceuticalsintended to modify directly the action of that protein. Thus, if a testligand is shown, through use of the present method, to bind a proteinthat reflects or affects the etiology of a condition, it may indicatethe potential ability of the test ligand to alter protein function andto be an effective pharmaceutical or lead compound for the developmentof such a pharmaceutical. Alternatively, the ligand may serve as thebasis for the construction of hybrid compounds containing an additionalcomponent that has the potential to alter the protein's function. Forexample, a known compound that inhibits the activity of a family ofrelated enzymes may be rendered specific to one member of the family byconjugation of the known compound to a ligand, identified by the methodsof the present invention, that binds specifically to that member at adifferent site than that recognized by the known compound.

The fact that the present method is based on physicochemical propertiescommon to most proteins gives it widespread application. The presentinvention can be applied to large-scale systematic high-throughputprocedures that allow a cost-effective screening of many thousands oftest ligands. Once a ligand has been identified by the methods of thepresent invention, it can be further analyzed in more detail using knownmethods specific to the particular target protein used. Also, the ligandcan be tested for its ability to influence, either positively ornegatively, a known biological activity of the target protein.

Referring now to FIG. 1, there is shown a plan view of one embodiment ofdetector 100 that is a part of the nanocalorimeter array in accordancewith the present invention. This example embodiment enables a passivethermal equilibration of the combined protein, water and ligand dropswith the device so that the resultant temperature changes can bedetected by means of a temperature sensing device. Because themeasurement region is kept small enough and sufficiently thermallyconductive, through the fabrication of a thermally conducting layer suchas aluminum or copper, the passive equilibration time can be made small.Suitable thermometer elements are based on thin film materials andinclude but are not limited to resistive thermometers, thermopiles andsurface acoustic wave devices (SAW). The preferred embodiment is basedon resistive thermometers made from thin film materials with a hightemperature coefficient of resistivity, for example amorphous silicon,Vanadium Oxide and Yttrium Barium Copper Oxide (YBCO).

Nanocalorimeter 100 includes thermal isolation layer 110, which containsmeasurement region 160 and reference region 170. Regions 160 and 170 mayalso be contained in separate isolation regions, as describedhereinbelow. Thermal isolation region 110 provides isolation fromsurrounding thermal environments, thus increasing measurement time andreducing thermal noise. Although layer 110 is used in this exampleembodiment to thermally isolate the reaction and temperature sensingcomponents of the nanocalorimeter 100, any means to thermally isolatethese components can be used in alternate embodiments of the presentinvention.

In this example embodiment, the thermal isolation layer 110 may comprisea plastic material in thin foil form (typically ranging from less than15 microns to approximately 25 microns in thickness for this embodiment,possibly as thin as 2 microns and as thick as 500 microns for someapplications). Candidate plastic materials include polyimide (forexample Dupont Kapton® and others), polyester (for example DupontMylar®) foil, PolyEtherEtherKetone (PEEK), or PolyPhenylene Sulphide(PPS). Alternatively, in embodiments, the thermal isolation regioncomprises other thin membranes of sufficiently low thermal conductivity,such as SiN and comparable materials.

Measurement region 160 and reference region 170 include thermalequilibrium regions 120 and 125 respectively, that are thermallyisolated from the detector's mechanical support. In this exampleembodiment, thermal equilibrium region 120 contains two resistivethermometers 140, which measure the reaction temperature, while thermalequilibrium region 125 contains a second set of two thermometers 140,which measure the variations in the background temperature. Theresistive thermometers are deposited in thermal equilibrium regions 120using standard fabrication techniques, including in embodiments, but notlimited to, lithographic patterning of thin films, micro-electronicfabrication techniques (e.g. including sputtering, chemical etching,evaporation), and printed circuit board fabrication techniques. Boththermal equilibrium regions 120 and 125 are sufficiently large toreceive and support separate drops of protein and ligand deposited bydirect printing and also to support the combination of these two dropsafter merging, triggered by an example drop merging device 130. Forexample, for a 400 nL final drop size, the detector, which includes themeasurement and reference regions, may be 3.7 mm by 4.6 mm. Each thermalequilibration region 120 and 125 has a sufficient thermal conduction forthe region to equilibrate quickly relative to the thermal dissipation.The regions have a sufficiently low heat capacity such that little ofthe heat of reaction is absorbed in the support. High thermalconductivity with low heat capacity may be accomplished, for example,with a metal film such as a 10 μm thick aluminum or copper filmextending over the area of the thermal equilibration region. In thisexample, for a 400 nL drop and a 10 μm thick aluminum film, the filmabsorbs approximately 7% of the heat of reaction.

As suggested above, the thermal equilibration regions must be thermallyisolated from their environment so that the temperature differencecaused by the reaction takes a relatively long time to dissipate. Thelonger this dissipation time, the longer the signal can be integratedduring measurement, which improves the signal to noise ratio. Forexample, a 10 second integration time corresponds to a 0.1 Hzmeasurement bandwidth and increases the signal to noise ratio by 3.2over a 1 second integration. Thermal dissipation occurs through fourdifferent channels: conduction across the supporting medium, conductionthrough the electrical interconnect, conduction through the surroundingenvironment and evaporation. For the example of conduction across thethermal isolation medium 110, the rate of heat transfer from the dropequals the thermal conductivity of the medium 110 multiplied by thecross section of the medium 110 through which the heat is conducted andthe temperature gradient across the region, orQ=ΛAdT/dx,where Λ is the thermal conductivity of the membrane, A is the crosssection of the region through which the heat is conducted and dT/dx isthe temperature gradient across the thermal isolation medium 110. NoteQ=C dT/dt where C is the heat capacity of the drop, and from thisT=T _(o) e ^(−ΛAt/CL),where t is the time, L is the length of the isolation region 110, alltemperatures are relative to the temperature of the surroundingenvironment, with the approximation dT/dx=T/L. The time constant, τ, forthermal dissipation is thereforeτ=CL/ΛA.

Consequently, the time constant increases with increases in the heatcapacity of the drop and decreases with increases in the rate of thermalconduction. Note that while the heat capacity of the drop increases withdrop size, increasing the drop size reduces the density of detectors onan array of detectors, increases the thermal equilibration time for thedrop, and uses valuable material. A lower array density means a largerarray size for a given detector number.

In the example embodiment, drop size is 400 nL for the combined dropafter merging. For this drop size, estimates of the time constantsassociated with different dissipation channels in the example embodimentare shown in the following table:

TABLE 1 Time Conduction across thermal isolation layer + interconnectleads 110 sec Conduction through vapor (Xe)  19 sec Evaporation (5° C.operation)  25 sec

For the purposes of the table, it was assumed that the thermal isolationlayer is 7 μm thick plastic and there are eleven interconnect leads withthickness of 0.1 μm for each thermal equilibrium region. As mentionedabove, the thermal isolation layer for this example embodiment may befabricated of a plastic material in thin foil form (typically rangingfrom less than 15 microns to approximately 25 microns in thickness forthis embodiment, possibly as thin as 2 microns and as thick as 500microns for some applications), thereby ensuring that the above timeconstant for conduction across the thermal isolation layer is largecompared with the measurement bandwidth. Examples of candidate plasticmaterials include polyimide (for example Dupont Kapton® and others),polyester (for example Dupont Mylar®) foil, PolyEtherEtherKetone (PEEK),PolyPhenylene Sulphide (PPS), polyethylene, or polypropylene. In theexample embodiment, the same material is; also used as the support forthe thermal equilibration region, including the resistive thermometers.

In the example embodiment, the same material may be used for the supportand the thermal equilibration, including the resistive thermometers.Consequently, one important consideration in selecting a substratepolymer is the highest temperature that is needed in subsequentdeposition and processing of thermometer, conductor and insulator filmsin the particular embodiment. As an example, the temperature needed inthe deposition of amorphous silicon thermometer material is typically inthe range of 170–250° C. This requires the selection of a substratepolymer film with a high softening temperature. These polymers mayinclude, but are not limited to, polyimide (PI), PolyEtherEtherKetone(PEEK), or PolyPhenylene Sulphide (PPS). Conversely, deposition ofVanadium Oxide thermometer material can be done at a substantially lowertemperature. This allows the selection of substrate polymers with alower softening point, such as Polyester (Dupont Mylar®).

These plastic substrates enable low cost manufacturing that can scale tolarge arrays of detectors, which enable fast and cost effective testingof large numbers of reactions. This invention anticipates, for example,detector array sizes of 96, 384, 1536 and larger. The low-cost detectorarrays might also be used once and then discarded, eliminatingtime-consuming washing steps and reducing problems withcross-contamination.

Another thermal consideration is the characteristic time for a drop toequilibrate with the detector after it is placed on the detector. Thisis a combination of the characteristic time for conduction of heatthrough the drop, t₁, and the characteristic conduction time across thedetector, t₂. In an example embodiment, an aluminum film is used toincrease the thermal conduction across the detector. An estimate of thecharacteristic time t₁ ist ₁=0.44R ²/α=0.61 sec,where R is the drop radius, in this example 460 μm, and α is the thermaldiffusivity of the drop, 0.0015 cm²/sec for water. For thin plasticsubstrates, the characteristic time for lateral conduction across thedetector is governed by conduction across the metal film incorporatedinto the design for temperature equilibration, which is an aluminumstrip in this example. An estimate for this characteristic time ist ₂=(ρC _(p) V)_(drop) ×L _(Al)/4R _(drop) ×δ×k _(Al)=0.44 sec,where ρ is the density of the drop, 1 g/cm³ in this example, C_(P) isthe specific heat, 1 cal/g° C. in this example, L_(Al) is the length ofthe conduction path along the aluminum strip from one drop to the other,2.5 R_(drop) in this example, δ is the aluminum strip thickness, 10 μmin this example, and k_(Al) is the aluminum thermal conductivity, 0.57Cal/C-Cm-sec. The aluminum thickness is selected to provide sufficientthermal conduction without contributing significantly to the heatcapacity of the detector. Heat capacity of the detector must be madesufficiently low so as to minimize the absorption of heat released fromthe reaction in the drop in order to minimize attenuation of thetemperature change arising from the reaction.

Each thermal equilibration region 120 and 125 contains thermometers 140and drop merging electrodes 130. Although for the purposes hereinthermometers 140 are shown spaced apart from more centrally-positioneddrop merging electrodes 130 on each thermal equilibration region 120 and125, this configuration is for means of example only. Provided that thedrop merging device 130 and thermometers 140 are in good thermal contactwith the high conductance film, the exact placement of thermometers 140and drop merging electrodes 130 is not important for thermalconsiderations.

In operation, the two resistive thermometers 140 situated in thermalequilibration region 120 detect the heat of reaction between anarbitrary protein and a ligand at low concentrations deposited withinthermal equilibration region 120. In this example, the heat of reactionis detected through measurement of a voltage change in a bridge circuitdue to the resistance change in the thermometers which are configured inthe bridge circuit. Resistive thermometers 140 in thermal equilibriumregion 120 detect a reaction between a sample ligand and a protein; theother resistive thermometers 145 in thermal equilibrium region 125 serveas a reference. Because the temperature rise due to the reaction may besmall, for example approximately 10 μ° C. for protein and ligandconcentrations of 1 μM and a heat of reaction of 10⁴ cal/mol, theresistive thermometers 140 are fabricated from materials that provide alarge change in resistance for a small temperature change.

In this example embodiment, the resistive thermometers 140 arefabricated from materials with a high temperature coefficient ofresistance, such as amorphous silicon Vanadium Oxide and Yttrium BariumCopper Oxide (YBCO). Similar small drops of non-reactive solution (forexample water or mixtures of water and DMSO) and target protein, thecontrol combination, are deposited close together in thermal equilibriumregion 125. Resistive thermometers 140 are configured as an AC bridgerepresented by AC generator 180 and ground 190, discussed in more detailhereinbelow. At a specified time after the drops have reached thermalequilibrium, they are moved together to initiate the reaction. Themovement operation creates sufficient mixing of the two drops in a timesmall compared to the measurement time. The heat released by theprotein-ligand reaction of the test combination causes a change in theresistance of the affected thermometers relative to the referencethermometers. This change in resistance causes the voltage at the:detection point to change from zero. This change is detected bysensitive, noise rejecting circuits such as a lock-in amplifier.

Referring now to FIG. 2, there is shown another example embodiment ofthe present invention. In this example embodiment, nanocalorimeter 200includes thermal isolation layer 210, which contains thermal equilibriumregion 220. Thermal isolation region 210 provides isolation fromsurrounding thermal environments, thus increasing measurement time andreducing thermal noise. In this example embodiment, thermal equilibriumregion 220 contains one resistive thermometer 240, which measures thereaction temperature. The resistive thermometer is deposited in thermalequilibrium region 220 using standard fabrication techniques, includingin embodiments, but not limited to, lithographic patterning of thinfilms, micro-electronic fabrication techniques (e.g. includingsputtering, chemical etching, evaporation), and printed circuit boardfabrication techniques. Thermal equilibrium region 220 is sufficientlylarge to receive and support separate drops of protein and liganddeposited by direct printing and also to support the combination ofthese two drops after merging, triggered by drop merging device 230.Thermal equilibration region 220 has a sufficient thermal conduction forthe region to equilibrate quickly relative to the thermal dissipation.The region also has a sufficiently low heat capacity such that little ofthe heat of reaction is absorbed in the support. High thermalconductivity with low heat capacity may be accomplished, for example,with a metal film such as a 10 μm thick aluminum or copper film.

Thermal equilibration region 220 contains thermometer 240 and dropmerging device 230. Although for the purposes herein thermometer 240 isshown spaced apart from more centrally-positioned drop merging device230 on thermal equilibration region 220, this configuration is for meansof example only. Provided that the drop merging device 230 andthermometer 240 are in good thermal contact with the high conductancefilm, the exact placement of thermometer 240 and drop merging device 230is not important for thermal considerations.

In operation, the resistive thermometer 240 situated in thermalequilibration region 220 detects the heat of reaction between anarbitrary protein and a ligand at low concentrations deposited withinthermal equilibration region 220. In this example, the heat of reactionis detected through measurement of a voltage change in a bridge circuitdue to the resistance change in the thermometer, which is configured inthe bridge circuit. Resistive thermometer 240 in thermal equilibriumregion 220 detects a reaction between a sample ligand and a protein.Because the temperature rise due to the reaction may be small, forexample approximately 1 m° C. for this embodiment, resistive thermometer240 is fabricated from materials that provide a large change inresistance for a small temperature change. In this example embodiment,resistive thermometer 240 is made of a material with a high temperaturecoefficient for resistance such as amorphous silicon, Vanadium Oxide andYttrium Barium Copper Oxide (YBCO).

Resistive thermometer 240 is configured as an AC bridge represented bydetection electronics 250, discussed in more detail hereinbelow. Theother three legs of the AC bridge are made of low temperaturecoefficient resistors located on the amplifier printed circuit board. Ata specified time after the drops have reached thermal equilibrium, theyare moved together to initiate the reaction. The movement operationcreates sufficient mixing of the two drops in a time small compared tothe measurement time. The heat released by the protein-ligand reactionof the test combination causes a change in the resistance of thermometer240. This change in resistance causes voltage at a detection point tochange from zero. This change is detected by sensitive, noise rejectingcircuits such as a lock-in amplifier. Alternatively if the reactions tobe measured produce enough heat, the resistance change of thethermometer may be measured by a direct DC resistance measurement.

Referring now to FIG. 3, there is shown another example embodiment ofthe present invention. In this example embodiment, nanocalorimeter 300includes thermal isolation layer 310, which contains thermalequilibration region 320. Thermal isolation region 310 providesisolation from surrounding thermal environments, thus increasingmeasurement time and reducing thermal noise. In this example embodiment,thermal equilibration region 320 contains two resistive thermometers340, which measure the reaction temperature. Each resistive thermometeris deposited in thermal equilibration region 320 using standardfabrication techniques, including in embodiments, but not limited to,lithographic patterning of thin films, micro-electronic fabricationtechniques (e.g. including sputtering, chemical etching, evaporation),and printed circuit board fabrication techniques. Thermal equilibrationregion 320 is sufficiently large to receive and support separate dropsof protein and ligand deposited by direct printing and also to supportthe combination of these two drops after merging, triggered by dropmerging device 330. Thermal equilibration region 320 has a sufficientthermal conduction for the region to equilibrate quickly relative to thethermal dissipation. The region also has a sufficiently low heatcapacity such that little of the heat of reaction is absorbed in thesupport. High thermal conductivity with low heat capacity may beaccomplished, for example, with a metal film such as a 10 μm thickaluminum or copper film.

Thermal equilibration region 320 contains thermometers 340 and dropmerging device 330. Although for the purposes herein thermometers 340are shown spaced apart from more centrally-positioned drop mergingdevice 330 on thermal equilibration region 320, this configuration isfor means of example only. Provided that the drop merging device 330 andthermometers 340 are in good thermal contact with the high conductancefilm, the exact placement of thermometers 340 and drop merging device330 is not important for thermal considerations.

In operation, two resistive thermometers 340 situated in thermalequilibration region 320 detect the heat of reaction between anarbitrary protein and a ligand at low concentrations deposited withinthermal equilibration region 320. In this example, the heat of reactionis detected through measurement of a voltage change in a bridge circuitdue to the resistance change in the thermometers, which are configuredin the bridge circuit. Resistive thermometer 340 in thermal equilibriumregion 320 detects a reaction between a sample ligand and a protein.Because the temperature rise due to the reaction may be small, forexample approximately 10 μ° C. for protein and ligand concentrations of1 μM and a heat of reaction of 10⁴ cal/mol, resistive thermometers 340are fabricated from materials that provide a large change in resistancefor a small temperature change. In this example embodiment, resistivethermometers 340 are made of materials with a high temperaturecoefficient for resistance such as amorphous silicon. Vanadium Oxide andYttrium Barium Copper Oxide (YBCO).

Resistive thermometer 340 are configured as an AC bridge represented bydetection electronics 350, discussed in more detail hereinbelow. Theother two legs of the AC bridge are made of low temperature coefficientresistors located on the amplifier printed circuit board. At a specifiedtime after the drops have reached thermal equilibrium, they are movedtogether to initiate the reaction. The movement operation createssufficient mixing of the two drops in a time small compared to themeasurement time. The heat released by the protein-ligand reaction ofthe test combination causes a change in the resistance of thermometers340. This change in resistance causes voltage at a detection point tochange from zero. This change is detected by sensitive, noise rejectingcircuits such as a Lock-in amplifier. Alternatively, if the reactions tobe measured produce enough heat, the resistance change of thethermometer may be measured by direct DC resistance measurement of thetwo thermometers connected in series.

Referring now to FIG. 4, there is shown yet another example embodimentof the present invention. In this example embodiment, nanocalorimeter400 includes thermal isolation layers 410, each of which contains athermal equilibration region. Thermal isolation regions 410 provideisolation from surrounding thermal environments, thus increasingmeasurement time and reducing thermal noise. In this example embodiment,thermal equilibration regions 420 and 425 each contain two resistivethermometers 440, which measure the reaction temperature. Each resistivethermometer is deposited in thermal equilibration regions 420 and 425using standard fabrication techniques, including in embodiments, but notlimited to, lithographic patterning of thin films, micro-electronicfabrication techniques (e.g. including sputtering, chemical etching,evaporation), and printed circuit board fabrication techniques. Thermalequilibration regions 420 and 425 are sufficiently large to receive andsupport separate drops of protein and ligand deposited by directprinting and also to support the combination of these two drops aftermerging, triggered by drop merging device 430. Thermal equilibrationregions 420 and 425; have a sufficient thermal conduction for the regionto equilibrate quickly relative to the thermal dissipation. The regionsalso have a sufficiently low heat capacity such that little of the heatof reaction is absorbed in the support. High thermal conductivity withlow heat capacity may be accomplished, for example, with a metal filmsuch as a 10 μm thick aluminum or copper film.

Thermal equilibration regions 420 and 425 contain thermometers 440 anddrop merging devices 430. Although for the purposes herein thermometers440 are shown spaced apart from more centrally-positioned drop mergingdevice 430 on thermal equilibration regions 420 and 425, thisconfiguration is for means of example only. Provided that the dropmerging devices 430 and thermometers 440 are in good thermal contactwith the high conductance film, the exact placement of thermometers 440and drop merging devices 430 is not important for thermalconsiderations.

In operation, the two resistive thermometers 440 situated in thermalequilibration regions 420 and 425 detect the heat of reaction between anarbitrary protein and a ligand at low concentrations deposited withinthermal equilibration regions 420 and 425. The two resistivethermometers 440 situated in thermal equilibration region 425 detect thetemperature of drops deposited and merged within thermal equilibrationregion 420. In this example, the heat of reaction is detected throughmeasurement of a voltage change in a bridge circuit due to theresistance change in the thermometers which are configured in the bridgecircuit. Resistive thermometers 440 in thermal equilibration region 420detect a reaction between a sample ligand and a protein; the otherresistive thermometers 440 in thermal equilibration region 425 serve asa reference. Because the temperature rise due to the reaction may besmall, for example approximately 10 μ° C. for protein and ligandconcentrations of 1 μM and a heat of reaction of 10⁴ cal/mol, theresistive thermometers 440 are fabricated from materials that provide alarge change in resistance for a small temperature change. In thisexample embodiment, the resistive thermometers 440 are made of materialswith a high temperature coefficient for resistance such as amorphoussilicon, Vanadium Oxide, and Yttrium Barium Copper Oxide (YBCO). Similarsmall drops of non-reactive solution (for example water or mixtures ofwater and DMSO) and target protein, the control combination, aredeposited close together in thermal equilibrium region 425.

Resistive thermometers 440 are configured as an AC bridge represented byAC generator 480 and ground 490, discussed in more detail hereinbelow.At a specified time after the drops have reached thermal equilibrium,they are moved together to initiate the reaction. The movement operationcreates sufficient mixing of the two drops in a time small compared tothe measurement time. The heat released by the protein-ligand reactionof the test combination causes a change in the resistance of theaffected thermometers. This change in resistance causes the voltage atthe detection point to change from zero. This change is detected bysensitive, noise rejecting circuits, for example a lock-in amplifier.

Referring now to FIG. 5, thermometers 510, 520, 530 and 540 form thefour resistive legs of one example configuration for a bridge circuitaccording to the present invention. Resistive thermometerssimultaneously measure temperature changes due to both the reaction andthe background drift. In this example, two measurement thermometers 530and 540 measure the reaction and two reference thermometers 510 and 520measure the background temperature changes. If the resistance of themeasurement thermometers changes, as happens when the temperature in themeasurement region increases, then the voltage at point R in the bridgebecomes more positive or negative relative to ground, depending on thepolarity of the voltage placed across the bridge circuit and the sign ofthe thermal coefficient of resistance, while the voltage at point L inthe bridge does the opposite, that is, becomes less positive or negativerelative to ground, respectively. This configuration maximizes thevoltage difference across detection electronics 550. As will beappreciated by one skilled in the art, other bridge configurations arepossible, such as one in which thermometer 540 has a low temperaturesensitivity and is not fabricated on the device and where 520 is avariable resistor used to balance the bridge and is also not fabricatedon the device.

Resistance thermometers 510, 520, 530 and 540 may be fabricated frompatterned thin film and are connected as a bridge. The resistance ofeach thermometer varies with temperature by an amount proportional tothe thermal coefficient of resistance of the material used. Sinceα=1/R ΔR/ΔT,it follows thatΔR=αRΔT,where R is resistance, T is temperature, and α is the thermalcoefficient of resistance of the thermometer material. Therefore, thesignal voltage across the resistor varies by

${{\Delta\; V_{S}} = {{\Delta\;{RI}} = {\alpha\; R\;\Delta\; T\sqrt{\frac{P}{R}}}}},$where V_(S) is the signal voltage, I is the current through theresistor, and P is power. The thermal noise in each resistor becomesV _(N)=√{square root over (4kTRB)}=1.2×10⁻¹⁰ √{square root over (RB)}where B is the measurement bandwidth in seconds, R is the resistance inOhms, and k is Boltzmann's constant. Assuming the detection system canbe constructed without introducing noise in excess of the thermal noise,the signal to noise ratio becomesS/N=8.3×10⁹ αΔT√{square root over (P/B)}.

Protein-ligand reactions are generally investigated at lowconcentrations during high-throughput screening, typically in the rangeof 10⁻⁵ to 10⁻⁶ M. The reactions typically release a heat of reaction,Q, which is on the order of 10⁴ Cal/mole. For illustrative purposes,consider combining 2 drops with concentrations of 2 μM of protein andligand, respectively. If the drops have equal volumes, the combinationhas a 1 μM concentration of each reactant. Additionally,CVΔT=MVQ,where V is the solution volume, C is the heat capacity of the solution,and M is the concentration in the mixed drop. Therefore,ΔT=MQ/C=10⁻⁶ mole/L×10⁴ Cal/mole/10³ Cal/° C.−L=10⁻⁵° C.,where Q is the heat of reaction, C is the heat capacity of the solute,and M is the concentration in the mixed drop.

For example, for a thin film thermometer made from a-Si., for whichα=2.8×10⁻²° C.⁻¹, and a bandwidth of 0.1 Hz, a signal to noise ratio of7 is achieved with 1 μW of power dissipated in the resistor. The voltagechange then becomesΔV _(S)=2ΔRI=2αΔTRI=4×10⁻⁷ RI=4×10⁻⁷ √{square root over (PR)}.

The following table provides the signal strength for various exemplarycombinations of thermometer impedance and power:

TABLE 2 Thermometer Detection Power Impedance Voltage S:N 1 μW 100 kΩ126 nV  7:1 1 μW   1 MΩ 400 nV  7:1 4 μW 100 kΩ 252 nV 15:1 4 μW   1 MΩ800 nV 15:1

The values in the table assume 1 μM concentrations of protein and ligandin the solution of the merged drops, 10⁴ cal/mol heat of reaction, and alarge enough binding constant such that almost all protein and ligandreact, resulting in the 10 μ° C. temperature rise cited above.

To initiate a reaction, the deposited drops need to be merged togetherand the drop contents well mixed. It is noted that numerous methods fordrop deposition are known in the art, any of which may operatebeneficially with the present invention for the purpose of dispersingdrops.

Although numerous means and methods for merging the deposited drops maybe utilized, for the purposes herein, one example method, the methodsdisclosed in copending U.S. patent application Ser. No. XX/XXXX(“Apparatus and Method for Using Electrostatic Force to Cause FluidMovement”), will be briefly described. To reduce complexity of thesystem and to increase reliability, this example drop merging methodutilizes electrostatic forces generated by a planar configuration of twoelectrodes to merge the two drops and cause equilibration through fastmixing. The electrodes are constructed from thin conducting films on thesurface of the device, using standard fabrication techniques, includingin embodiments, but not limited to, lithographic patterning of thinfilms, micro-electronic fabrication techniques (e.g. includingsputtering, chemical etching, evaporation), and printed circuit boardfabrication techniques.

Referring now to FIG. 6, the merging electrodes are formed fromconducting film 620 and the conducting film 610, which are positioned onthe surface of substrate 650 and covered by insulating layer 640. Inthis example, conducting films 610 and 620 may be approximately 1.0 mmby 0.8 mm in size, with a thickness ranging in size from approximately0.1 μm to approximately 10 μm, and are separated by a gap ofapproximately 50 μm and are made of aluminum or copper thin film; theinsulating layer may be approximately 0.1 μm to approximately 2 μm inthickness and may, for example, be made of silicon oxide or siliconnitride or silicon ox, nitride, or spin-, spray-, or otherwise depositedpolymers, such as parylene, Dupont Teflon AF, 3M Fluorad products, 3MEGC 1700, other fluoropolymers, polysiloxanes, diamond-like carbon orother spin-coated, spray-coated, dip coated, or vapor depositedpolymers. Suitable insulator materials have a high electricalresistivity, chemical & mechanical durability and have no pinholes indeposited thin film form. For illustrative purposes, also shown is highconductance film 680, that enables thermal equilibration in the thermalequilibration region. Protein drop 660 is deposited asymmetricallyacross the surface above conducting films 610 and 620 such that the dropdisproportionately occupies the surface above one of the conductingfilms. In this example, 93% of protein drop 660 occupies the surface onthe side of meridian 670 above conducting film 620 and 7% of proteindrop 660 occupies the surface on the side of meridian 670 aboveconducting film 610.

Ligand drop 630 is deposited on the surface above conducting film 610.When a voltage is applied, preferably in the form of a voltage pulse,across conducting films 610 and 620, drop 660 is propelled towardstationary drop 630 and the drops merge. While the comparative dropsizes of protein drop 660 and ligand drop 630 may be equal, unequal dropsizes may also be used. The hydrophobic surface of insulating layer 640minimizes the adhesion of drops 630 and 660 to the surface, whichreduces the drag on the drops during merging. In this example, thehydrophobic surface is made of a fluorinated polymer, such as, forexample, 3M Fluorad, Dupont Teflon AF, 3M EGC-1700, or plasma-depositedfluorocarbons. In one embodiment, a Parylene coating may be used as theinsulator layer, as well as for the hydrophobic surface.

Alternatively, in an alternate embodiment, the thermometer material(e.g. amorphous silicon) itself may be utilized to construct drop moverelectrodes. In another embodiment, the electrodes and thermometer may befabricated in different layers, with the electrodes in a layer betweenthe drop deposition points and the thermometer, to enable placing metaldrop mover electrodes on top of the thermometers. In this embodiment, anelectrically insulating layer separates the thermometers and electrodes.

Several technologies are available for drop delivery, with one of thesetechnologies being syringes. For example, Hydra Microdispensers (made byRobbins Scientific, Sunnyvale, Calif.) dispense liquids into all wellsof 96 or 384 microplates simultaneously. As stated by Robbins Scientificin their product information, “The barrels of 96 or 384 syringes areheld in a fixed array centered along an X-Y grid that corresponds to theexact center of each well in a microplate. The plungers within thesyringes move up and down under computer control, dispensing oraspirating liquids from microplates to the detector array. A precisionmotor assembly smoothly moves the plungers in increments of 2.5 microns.When equipped with 100 μl syringes, this allows accurate dispensing ofvolumes as low as 100 nl. For the purposes herein, such a system couldbe utilized to deliver, for example, 100 nL ligand and 300 nL proteindrops.

Cartesian Technologies provides a syringe-based system for deliveringdrops, wherein the Cartesian system utilizes syringe-syringepump-solenoid assemblies to accurately aspirate and dispense drops from20 nL to 250 μL, which is a range sufficient to satisfy the drop sizerequirement of the present embodiment.

Packard BioSciences markets a system with piezoelectric-driven tips foraspirating liquids and delivering drops. The BioChip Arrayer product isa system with four dispensers (as compared with the full 96 or 384 ofthe Hydra Dispenser discussed above), and it delivers 300 nL in 2seconds. If needed, several of these units may be used in parallel tosatisfy the requirements of the present invention. Conversely, forembodiments with a much smaller numbers of samples, one unit may besufficient.

Pin spotting is a technology commonly used to place drops of solutiononto slides in the DNA MicroArray industry. Pin spotting is often usedfor drops much smaller than 100–300 nL, as is common in the DNAMicroArray industry. However, there are some pin spotting technologiesthat can deliver drops with volumes in the range of 100 nL to 500 nL, asis preferred in an example embodiment of the present invention. Forexample, V&P Scientific sells pin spotting solutions for drops in thissize range.

Referring now to FIG. 7, there is shown a schematic of one exampleembodiment of the electronic measuring system utilized herein. For thepurposes of example, an alternating current (AC) detection method isillustrated. The AC detection method eliminates the 1/f noise inherentin electronic devices, particularly the a-Si thermometer, in which the1/f noise can be significant at frequencies up to 1 kHz. A bridgecircuit is used to detect changes in the resistance of the thermometers.The electronics implements four functions: amplification of the outputof the bridge, zeroing of the bridge, detection of the signal, andcomputer analysis of the signal. For each bridge 710, a sine wave isprovided by generator 780. This sine wave drives the two input terminalsof each bridge.

Each bridge has two output terminals whose difference represents thetemperature difference of the reference and measurement cells of thebridge. The signal on these two terminals is amplified by a low-noisesignal amplifier 720. Because the signal level is low, noise introducedby this function must be minimal, but noise minimization must bebalanced by design considerations. For example, for the array to bedisposable, which is desirable in some applications, the amplifiers mustbe located off the array, but amplifiers placed on the periphery resultin the introduction of noise through the longer lead length. To minimizenoise from interconnect, the amplifiers may be placed on a separatetemperature-controlled heat sink positioned in close proximity to thedetector array, with amplifiers 720 placed directly above the detectorarray and contacting the array through compressible pogo-pin connectors.An additional advantage of placing each amplifier directly above itsassociated bridge is that the bridge output signal wires do not have topass near any other wires thereby reducing noise coupling.

A multiplexor 730 enables several individual detectors to use eachlock-in amplifier and digitizer. With the embodiment of the inventionshown in FIG. 7, advantage is derived by the use of one amplifier foreach detector and placement of the multiplexor 730 after the amplifier.The noise introduced by the multiplexor contributes a smaller relativeamount than if the multiplexor had teen placed before the signalamplifier. Alternatively, if noise levels permit, the multiplexor couldbe placed before the signal amplifiers, allowing fewer signal amplifiersand a more compact arrangement of amplifiers and bridges.

The temperature sensors in each bridge may be similar but not identicalwith each other. After temperature equilibration, the output of thebridge will not quite be zero because of these differences. The outputwill be a small sine-wave proportional to the difference. This commonmode signal, if not reduced, limits the amount of amplification betweenthe bridge and the lock-in amplifier mixer. This in turn limits thesystem sensitivity. This common mode signal is minimized by use of thebridge zero operation that is performed after the initial amplificationthrough second stage amplifier 740 through offset voltage 750. A controlsignal selects a proportion of the sine-wave reference signal to besubtracted out of the amplified input signal. This control signal is setby measuring the output after equilibration and set its value tominimize the common mode output. If the inherent balance of the bridgeis sufficient, then the offset amplifier is not needed.

A lock-in operation 760 produces a dc output equal to the amplitude ofthe detector signal and may be implemented through known electroniccircuitry. A standard lock-in amplifier known in the art contains anamplifier that increases input signal amplitude. The signal is thenfiltered by a bandpass filter with a center frequency the same as areference sine-wave to remove noise at frequencies other than thereference frequency. A reference sine-wave is also input to the circuit.It is converted to a sine-wave and its phase is shifted to correspond tothe phase of the input signal. The reference signal is then mixed withthe input signal to create a composite output signal with alow-frequency component that represents the signal and a high-frequencycomponent that represents the noise. A low-pass filter removes thehigh-frequency noise component. Alternatively, the lock-in operationcould be implemented in software. The output of the lock-in operation isdigitized by A-D converter 770 and input into computer 790 for analysis.The amplitude of this digitized signal represents the temperaturedifference on the bridge. After the drops are moved together and when areaction occurs, its amplitude will increase until the drops are fullymixed and then decrease as heat is removed through conduction andevaporation. If no reaction occurs, no significant change will occur inthe signal. The computer correlates the digitized signal against theexpected temperature increase and decrease. If the correlation ispositive, then the occurrence of a reaction is signaled.

Referring now to FIG. 8, in one embodiment of the present invention, thedetectors of the detector array are arranged in a rectilinearorientation to form a matrix array. In this example, the array isfabricated on thin plastic sheet 810, for example a 10 μm thick Kapton®plastic substrate, and is supported by heat sink 820, which is made of amaterial with a high thermal conductivity such as Cu or Al. Thin filmconducting lines 850 placed in the regions between individual detectors830 serve as electrical interconnect that carry signal and power betweenthe detectors and the electronic module on the outside. Detectors 830require interconnect for the signal excitation and the drop mergingelectrodes. All detectors in pairs of adjacent rows are connected tocommon merge-electrode power 880. In embodiments, the resistivethermometers, drop merging electrodes, and electrical interconnect maybe patterned on one side of the matrix array, and the thermalequilibration film may be fabricated on the other side. In anembodiment, measurements are made simultaneously in two rows. Detectorsignal and ground are provided through contact pads located over theheat sink adjacent to each detector and connected to the array throughdetector amp contact pads 840. Common bridge-excitation is provided forpairs of rows by bridge power conducting lines 870. The merge-electrodepower and common bridge-excitation are introduced through alternatingrows. Because it is desirable to transfer fluids from standard storagedevices, such as well-plates having different densities (96 well, 384well, or 1536 well) the detectors have the same 9 mm square layout asstandard 96 well-plates used in the biotechnology and pharmaceuticalindustries.

Referring now to FIG. 9, there is shown an example cross-section of thenanocalorimeter assembly and its detector environment, which providesthermal isolation, electrical connections and sample delivery. Toachieve thermal isolation, the environment is structured to insure thatthe heat transferred to or from the drop is inconsequential. The threemain heat transfer channels for the assembly include: thermal conductionthrough the air, thermal conduction across the supporting medium, andevaporation. In embodiments, measurements can be conducted at lowtemperatures and high humidities, for example 5° C. in near 100%relative humidity (preferably noncondensing), to reduce evaporation toacceptable limits. Specifically, evaporation is controlled in part bymaintaining near 100% relative humidity, within some acceptabletolerance, of the solvent used to dissolve the chemicals beinginvestigated. This may be accomplished by exposing a reservoir ofsolvent to the atmosphere in the chamber enclosing the detector. Thelower temperature reduces the vapor pressure of the solvent, and higherhumidities reduce the concentration gradient of solvent in the gas phasenear the surface of the drop, thereby reducing the driving force forevaporation.

In other embodiments, reasonable measurements might be attainable athigher temperatures or higher humidities despite the correspondinglyhigher evaporation rates, in which cases operation at low temperaturesor high humidities may not be necessary. Thermal conductivity throughthe surrounding environment can be reduced in embodiments through use ofa controlled atmosphere, for example an environment rich in Xenon orArgon, which have lower thermal conductivities than air. In embodiments,conductivity can also be controlled through the use of a partial orcomplete vacuum, aerogels or other insulating materials, and othermethods that will occur to those skilled in the art.

To minimize thermal conduction across the supporting medium, detector910 resides on substrate 915, which is supported by substrate carrier925, which is in contact with heat sink 920. In this example heat sink920 is comprised of copper, but other materials known in the art couldalso be utilized. In embodiments heat sink 920 may be in thermal contactwith an optional active temperature control device 930, which controlsthe temperature of the heat sink to within 1 m° C. to 0.1° C. ofamplifier heat sink 940. The detector amplifiers dissipate power (10 mWearth), which may be too much heat for the detector heat sink in someembodiments. The amplifier power can be sunk to a separate heat sink ifdesired. Signal amplifiers 990 reside on amplifier printed circuit board950, which is in contact with heat sink 940. The temperature of heatsink 940 can be controlled by a temperature control device 960, ifdesired for a particular embodiment. Pogo pin connectors 980 connectamplifier printed circuit board 950 with detector substrate 915 throughamplifier pads 970. There are several conditions in which the heat sink920 does not need to be temperature controlled. In these cases, the heatsink is thermally isolated from the enclosing chamber using standard lowconduction materials like glass, plastic or stainless steel tubing. Inthese cases, the Amplifier PCB 950 is placed in direct contact with thetemperature controlled enclosing chamber.

To better appreciate the magnitude of temperature fluctuations that heatsink 920 may experience due to fluctuations of the chamber wall, theheat capacity for various copper heat sinks corresponding to variousarray sizes is shown in the table below. For the purposes of the table,all heat sinks are 3 cm in thickness. The heat sink area is shown belowfor the different examples, and the heat sink is assumed to be separatedby 10 cm from the chamber walls, with Xenon gas at about 0.1 to 2 atmfilling the space.

TABLE 3 Well Array Array Heat Sink Time Number Design Size Capacity AreaConstant 96 12 × 8  14.8 cm × 11.2 cm  1500 J/C  485  515000 cm cm² 38424 × 16 25.6 cm × 19.4 cm  4500 J/C 1263  594000 cm cm² 1536 48 × 3247.2 cm × 32.8 cm 14000 J/C 2030 1150000 cm cm²

As shown in the table, with the smallest time constant over 500,000seconds, a small 0.1° C. step-change temperature difference between thearray heat sink and the chamber wall would change the heat sink by 2 μ°C. over a 10 second measurement. Note that since the measurement isdifferential, as both the measurement and reference thermometers changeby the same amount providing common mode rejection, and since thechamber walls can be controlled to 0.1° C. with variations that are notrapid, the heat sink provides more than adequate temperature stabilityfrom conduction to the wall. Because the electrical connection to thedetector array increases the thermal conduction between the heat sinkand the chamber wall, conductance down the leads may be activelycontrolled in embodiments by placing the leads in thermal contact withan intermediate heat sink 945 whose temperature is actively controlledto be equal to that of the heat sink.

To better appreciate the magnitude of temperature fluctuations that heatsink 920 may experience due to heat conduction through the Pogo pins, anexample of Pogo pins contacting a 384-detector array is considered inthe table below. Pogo-pin connector 980 materials, for example brass,are selected for thermal performance. In this example, the powerconducted per pin for a 0.1 C. temperature difference across the pin isshown in the following table along with the total power conducted for a384-detector array (assuming three pins for each detector) for a 0.1° C.and a 0.1 m° C. temperature difference across the pin. For the purposesof the calculations shown in the table, a circular contact area with adiameter of 10 μm is assumed. To minimize the area of contact, the pogopins may be the pointed-tip type. The temperature change during a 10second measurement time is also shown. To keep the temperature changesufficiently low, the temperature of amplifier printed circuit board 950is accurately controlled in embodiments, either through active controlor by thermal contact with the chamber wall. For example, to limit thetemperature rise ΔT of heat sink 920 to 13 μ° C. or less over 10seconds, the values in Table 4 show that the temperature of theamplifier printed circuit board 950 must be controlled to within 0.01°C. Such control is possible using known active temperature controlelements, such as Peltier devices, a circulating-fluid refrigerationsystem, a heat pump, or any of a number of other activetemperature-control devices known by those skilled in the art.Alternatively, if the chamber wall is controlled to within 0.01° C.,then thermal contact of the amplifier printed circuit board with thechamber wall is sufficient to limit ΔT to 13 μ° C.

TABLE 4 Power/Pin Total Power AT in 10 sec Total ΔT in 10 sec (0.1 Ccontrol) (0.1 C control) (0.1 C control) (0.1 mC control) (0.1 mCcontrol)  1.2 cm pin 49.2 μW 56.6 mW 0.13 mC 56.6 μW 0.13 μC 0.56 cm pin47.7 μW 54.9 mW 0.12 mC 54.9 μW 0.12 μC

FIG. 10 illustrates a cross section of the measurement system utilizingthe array described herein. The measurement system in this exampleconfiguration includes two compartments, load lock chamber 1010 andmeasurement chamber 1050. The chambers and the atmosphere containedwithin them are equivalent; they are at the same operating temperature.The atmosphere within each chamber is a non-reactive gas, for examplexenon, argon, air, or nitrogen, at a near 100% relative humidity for thesolvents used in the drops being measured, and this humidity level ismaintained through use of vapor pressure reservoirs 1045. Thetemperature of the chamber walls is controlled to within 0.1° C. Heatsink 1085 receives heat from the power dissipated in the measurementthermometers on detector array 1040 when in the measurement chamber1050. In this example four thermometers are used for each detector, asshown in FIG. 1, and each thermometer dissipates approximately 4 μW. Therate of temperature increase of the heat sink due to thermometer heatingis approximately 10 μ° C. during a 10 second measurement, based on a 96detector array and a heat sink with a heat capacity of 1500 J/° C.(refer to Table 3 above). Detector array 1040 is connected to detectorarray electronics 1030 which in turn are connected to system electronics1060. Biomaterials are contained within a biomaterial storage well plate1015, which is placed in the load lock chamber 1010. In the measurementchamber 1050 are detector electronics 1075 as well as the associatedheat sink/controller 1070 for the detector electronics.

Biomaterials 1025 are deposited on the array with chemical depositiondevice 1035 in preparation for the measurement. While in the load lockchamber 1010, the heat sink 1085 and associated detector 1040 andbiomaterials 1025 are brought into thermal equilibrium with the chamberthrough heat conductor 1090. Heat conductor 1090 may be any material orsystem of high thermal conductivity, and may be, for example, a metalblock such as copper or aluminum that is in good thermal contact withboth the chamber wall and the heat sink 1085. As shown in FIG. 10,thermal contact of heat conductor 1090 with heat sink 1085 occursthrough the array transporter 1020. However, this configuration isexamplary only; other configurations will occur to those skilled in theart and are contemplated by the disclosure herein.

In alternative embodiments, heat conductor 1090 may have activetemperature control, such as control by a circulating-fluidrefrigeration or heating system, a Peltier device, a resistive heater, aheat pump, or any of a number of other active temperature-controldevices known by those skilled in the art. Furthermore, in alternativeembodiments the heat conductor and associated temperature controlfunction can be integrated into the array transporter 1020. Arraytransporter 1020 moves a detector array with deposited biomaterials fromthe load lock into measurement chamber 1050 and, in this example,utilizes a circular motion so that a detector array with measuredmaterials is simultaneously moved from the measurement chamber to theload lock. Other array transport methods may be utilized, such aspick-and-place devices and belt devices with elevators.

Once in the measurement chamber, the detector array is raised intocontact with the Pogo pins, and simultaneously the heat sink 1085 israised above the transporter 1020 and thermally isolated from it bysupporting pins 1053. The supporting pins may be fabricated from anygood thermal insulating material with sufficient mechanical strength,such as glass rods, stainless steel hollow tubing, plastic rods, porousceramics, and other materials known to those skilled in the art. Otherconfigurations are possible; for example, in alternative embodiments atemperature controller may be used to maintain heat sink 1085 at aspecified temperature in measurement chamber 1050, for example within 1m° C. of the temperature of heat sink/controller 1070 of detectorelectronics 1075, rather than relying on thermal isolation alone. Pogopin detector connectors 1080 make electrical contact directly to thedetectors to transmit thermal change information from the detector arrayto detector electronics 1075. This type of connector is used in thisexample to provide a nonpermanent connection that allows connection tobe made to successive arrays with low thermal contact to the array andgood placement accuracy with a small foot-print that providessymmetrical contact to the measurement and reference regions to enableprecise differential measurements.

In operation, detector array 1040, whose initial temperature is within1° C. of the temperature of load lock chamber 1010, is placed in loadlock chamber 1010 while a previously set-up detector array 1055 is beingmeasured in measurement chamber 1050. The proximity of measurementchamber 1050 to load lock chamber 1010 enables the connected detectorarray to be moved between the chambers while remaining in a controlledenvironment. Biomaterials are then moved into load lock chamber 1010 andstored in an appropriate vehicle 1015, such as a 384 or 1536 well plate,although other containers or well plate sizes would also be appropriate.Biomaterials 1025 are then deposited on detector array 1040 using, forexample, an aspirating/printing system or an automated syringe-typeloader 1035. Deposition device 1035 is maintained at a controlledtemperature to avoid warming biomaterials 1025. Initially, detectorarray 1040 is connected to detector array electronics 1030 and systemelectronics connector 1060, which provides the necessary electricalconnections to all the detector elements in detector array 1040 with theexception of the detector electronics for the measurement bridge.Depending on conditions, the detector bridges in detector array 1040 maybe driven by the AC sine wave (for example, element 560 in FIG. 5) toself-heat to a temperature that equilibrates the drop temperature withthe controlled environment in the load lock chamber. This signal isconducted through the system electronics connector 1060 to the detectorarray electronics 1030.

After the deposited materials 1025 come to thermal equilibrium with thedetector array 1040, the detector array 1040 with deposited chemicals1025 is then moved from load lock chamber 1010 to measurement chamber1050 by array transporter 1020 and measured detector array 1055 is movedinto load lock chamber 1010. This movement may be accomplished through arotation, such as a 180-degree rotation, or by any other means known inthe art. Within measurement chamber 1050, the detector array is inthermal contact with heat sink 1085, which in this embodiment isthermally isolated from transporter 1020 by supporting pins 1053 inmeasurement chamber 1050. The measurement sequence is initiated byapplying the AC-sine wave to the detector bridges. This signal iscreated by an AC generator located on the amplifier printed circuitboard 1075 and conducted to detector array 1055 through the pogo pins1080. The detector bridge is then zeroed by properly setting the offsetvoltage. Thermal equilibration is confirmed by measuring the voltageacross the detector bridge for a short period of time. When the rate ofchange: of this voltage is below a pre-specified level, the system is inthermal equilibrium. The zeroing operation may need to be repeatedduring this process.

A row of drops of deposited chemicals 1025 is then merged and mixed onthe surface of the detector array. This is accomplished by applying adrop moving voltage from the amplifier printed circuit board 1075through the pogo pins 1080 to the detector array 1055. The transientvoltages generated from the merging voltages are allowed to dissipate.The reaction during mixing is then measured by detecting the imbalancein the bridge. Each bridge in the row is measured repeatedly for aperiod of time and the data is input into the computer for analysis.

The individual bridges in a single row may be multiplexed in thedetection electronics. A measurement is made on one detector and thenthe next detector in the row until all the detectors in the row havebeen measured. This is repeated for a period of time until allmeasurements for the row are complete. Alternatively, multiple instancesof the detection electronics can simultaneously measure all the detectorarrays in the row. To further reduce measurement time, measurements maybe performed in blocks of two or more rows.

While the present invention has been illustrated and described withreference to specific embodiments, further modification and improvementswill occur to those skilled in the art. It is to be understood,therefore, that this invention is not limited to the particular formsillustrated and that it is intended in the appended claims to embraceall alternatives, modifications, and variations which do not depart fromthe spirit and scope of this invention.

1. A nanocalorimeter array for detecting chemical reactions thenanocalorimeter array comprising: a substrate; at least two thermalisolation regions residing on said substrate, wherein said thermalisolation regions comprises a material having low thermal conductivity;at least one reference region and at least one measurement regionresiding within said thermal isolation regions, wherein said at leastone reference region is operatively associated with said at least onemeasurement region, and wherein said reference region receives referencereagents; at least two drop merging electrodes residing within each saidreference region and each said measurement region for mixing solutionson the nanocalorimeter array, wherein drops of said solutions are mergedby application of a voltage to the electrodes; at least one thermalequilibration region positioned within each said reference region andeach said measurement region adjacent to and mechanically supported bysaid thermal isolation regions, wherein said drops of said solutions arebrought to thermal equilibrium before being merged; and at least onethermal measurement device residing as a layer within each said at leastone thermal equilibration region, wherein said thermal measurementdevice is connected to detection electronics.
 2. The nanocalorimeterarray according to claim 1, wherein a least two thermal measurementdevices reside within each of said thermal equilibration regions.
 3. Thenanocalorimeter array according to claim 1, wherein said thermalmeasurement device measures temperature change within each said thermalequilibration region, and wherein said detection electronics comparesaid measured temperatures for said at least one reference region andsaid at least one a measurement region, wherein said reference regionand said measurement region are physically separate from each other. 4.The nanocalorimeter array according to claim 1, wherein said at leastone thermal measurement device comprises a resistive thermometer.
 5. Thenanocalorimeter array according to claim 1, wherein said at least onethermal measurement device comprises a thermopile.
 6. Thenanocalorimeter array according to claim 1, further comprising means fordepositing chemical samples on said nanocalorimeter array.
 7. Thenanocalorimeter array according claim 1, wherein said drop mergingelectrodes reside on a first layer and said at least one thermalmeasurement device resides on a second layer with an insulating layerinterposed therein between, such that each said two drop mergingelectrodes are positioned adjacent to and physically touching saidthermal measurement device.
 8. The nanocalorimeter array according toclaim 1, wherein said drop merging electrodes comprise said thermalmeasurement device.
 9. The nanocalorimeter array according to claim 1,wherein said detection electronics comprise AC detection electronics.10. The nanocalorimeter array according to claim 1, wherein saiddetection electronics comprise a software program.
 11. Thenanocalorimeter array according to claim 1, further comprising means fordispensing chemical samples within specific locations on said thennalequalization regions.
 12. The nanocalorimeter array according to claim1, wherein said nanocalorimeter array further comprises a plurality ofthermal isolation regions residing on said substrate.
 13. Thenanocalorimeter array according to claim 1, wherein said substrate is inthermal contact with a first heat sink.
 14. The nanocalorimeter arrayaccording to claim 13, wherein said first heat sink is a temperaturecontrolled heat sink.
 15. The nanocalorimeter array according to claim1, wherein at least some of said detection electronics reside on aprinted circuit board in thermal contact with a secondtemperature-controlled heat sink.
 16. The nanocalonmeter array accordingto claim 4, wherein said resistive thermometer comprises amorphoussilicon.
 17. The nanocalorimeter array according to claim 4, whereinsaid resistive thermometer comprises silicon.
 18. The nanocalorimeterarray according to claim 4, wherein said resistive thermometer comprisesyttrium barium copper oxide.
 19. The nanocalorimeter array according toclaim 4, wherein said resistive thermometer comprises vanadium oxide.20. The nanocalorimeter array according to claim 4, wherein saidresistive thennometer comprises, mercury cadmium telluride.
 21. Thenanocalorimeter array according to claim 1, wherein said detectionelectronics comprise: at least one bridge; at least one signal amplifierfor amplifying bridge output; at least one multiplexer for selectingfrom a plurality of said signal amplifiers; and a lock-in circuit forproducing a DC output equal to the amplitude of the detector signal. 22.The nanocalorimeter array according to claim 1, further comprising adetecting environment.
 23. The nanocalorimeter array according to claim22, wherein said detecting environment provides thermal isolation,electrical connections, and delivery of chemical samples and comprises:at least one load lock chamber; and at least one measurement chamber.24. The nanocalorimeter array according to claim 23, wherein saiddetecting environment further comprises a temperature-controlledreservoir.
 25. The nanocalorimeter array according to claim 1, whereinsaid detection electronics are detachably connected to thenanocalorimeter array.
 26. The nanocalorimeter array according to claim25, wherein pogo pins detachably connect said detection electronics andthe nanocalorimeter array.
 27. The nanocalorimeter array according toclaim 1, wherein the chemical reactions comprise biochemical reactions.28. A nanocalorimeter array for detecting chemical reactions thenanocalorimeter array comprising: a substrate; at least two thermalisolation regions residing on said substrate, wherein said thermalisolation regions comprises a material having low thermal conductivity;at least one reference region and at least one measurement regionresiding within said thermal isolation regions, wherein said at leastone reference region is operatively associated with said at least onemeasurement region, and wherein said reference region receives referencereagents; at least one thermal equilibration region positioned withineach said reference region and each said measurement region adjacent toand mechanically supported by said thermal isolation regions, whereindrops of said solutions are brought to thermal equilibrium before beingmerged; and at least one thermal measurement device residing as a layerwithin each said at least one thermal equilibration region, wherein saidthermal measurement device is connected to detection electronics, andwherein said detection electronics are detachably connected to thenanocalorimeter array.